The present invention is concerned with a method for the preparation of an infectious IBDV mutant capable of replication in CEF cell culture, a genetically engineered IBDV mutant as well as with a vaccine such a IBDV mutant.
Infectious bursal disease virus (IBDV) IS a member of the Birnaviridae family. Viruses in this family have a very similar genomic organisation and a similar replication cycle. The genomes of these viruses consist of 2 segments (A and B) of double-stranded (ds) RNA. The larger segment A encodes a polyprotein which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4. VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of birnaviruses, and contains the antigenic regions responsible for the induction of neutralising antibodies. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. The larger segment A possesses also a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene. This second open reading frame encodes a protein VP5 of unknown function that is present in IBDV infected cells. The smaller segment B encodes VPI, a 90 kDa multifunctional protein with polymerase and capping enzyme activities.
For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes may be differentiated by virus neutralisation (VN) tests. Furthermore, subtypes of serotype 1 have been isolated. These so-called xe2x80x9cvariantxe2x80x9d viruses of serotype 1 can be identified by cross-neutralisation tests, a panel of monoclonal antibodies or RT-PCR. These subtypes of serotype 1 of IBDV have also been described in literature, for example: classical, variant-E, GLS, RS593 and DS326 strains (Van Loon, et al. Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 179-187, 1994).
Infectious Bursal disease (IBD), also called Gumboro disease, is an acute, highly-contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate mortality rates. Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius which is essential to the defence mechanism of the chicken. The IBD-virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
For many years the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine, to chickens that had been primed with attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevents early infection with IBDV and diminishes problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
Recently, very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe. The current vaccination programs failed to protect chicks sufficiently. Vaccination failures were mainly due to the inability of live vaccines to infect the birds before challenge with virulent field virus.
Therefore, a constant need exists to improve existing vaccines and to develop new types of vaccines. For the development of live vaccines IBD viruses in attenuated form are required. Conventionally, this can be achieved by serial passaging of IBDV field isolates on an appropriate substrate. For the development of inactivated IBDV vaccines, an appropriate substrate is necessary for the generation of high amounts of IBDV antigen mass resulting from the propagation of IBD viruses on the substrate.
It is known that field IBDVs can readily be propagated in vivo in the bursa of infected birds or in embryonated eggs. However, although, the successful adaptation an propagation of some IBDV strains to in vitro cell culture of chicken embryo origin has been reported, it is generally acknowledged that most IBDV strains isolated from infected bursa in the field, in particular the so-called virulent- or very virulent IBDV strains cannot be adapted to cells of chicken embryo origin, such as chicken embryo fibroblasts (CEF) or cells from other organs such as the kidney and liver (Brown et al., J. Gen. Virology 75, 675-680, 1994; van Loon, et al., 1994, supra).
The drawbacks of the in vivo culture substrates are obvious. Such culture methods are animal unfriendly, need a lot of animals, are time consuming and cannot be carried out under standardised and stringent conditions. In addition, the limited number of IBDV strains which are not refractory to adaptation to in vitro cell culture substrates, suffer from the disadvantage that as a result of the serial passaging process leading to the adaptation of the IBDV strains, random mutations are introduced in the genome of the virus in an uncontrolled manner. Such mutations may influence properties of the virus other than that associated with the adaptation of the virus to the cell culture, e.g. properties related to the immunogenicity of the virus. Such additional, random mutations are not desired. The adaptation of the IBDVs by passaging of the virus in vitro in CEF cell cultures has been associated with attenuation of the virulence as demonstrated by a reduction of the virus"" ability to induce lesions in the bursa of the infected bird. Yamaguchi et al. (Virology 223, 219-223, 1996) investigated the molecular basis for the virulence of IBD viruses and the attenuation of these viruses as a result of the adaptation of bursa IBDVs to CEF cell culture. It was concluded that from the studies carried out by Yamaguchi et al. the precise mutations involved in attenuation of the wild-type IBDV could not be identified. It was suggested that the amino acid residues at position 279 (Asp/Asn) and 284 (Ala/Thr) of the polyprotein encoded by the long open reading frame of the segment A are important for virulence or propagation of the IBDV in CEF cells. The latter was confirmed by Lim, B-L (Proceedings of the 4th Asia Pacific Poultry Health Conference, 22-26 November, 1998, Melbourne, Australia, Abst. 79). It is disclosed therein that substitution of the amino acid residues 279 (Aspxe2x86x92Asn) and 284 (Alaxe2x86x92Thr) in the VP2 protein of an IBDV results in a IBDV mutant which can be propagated in CEF cell culture. However, the prior art does not teach an alternative of type and minimal number of amino acid mutations which are required and sufficient to allow the adaptation of bursa IBDV to CEF cell culture.
It is an object of this invention to provide a generally applicable method for adaptation of IBDV isolates which only grow in vivo in the bursa of infected birds to growth in cell culture.
It is a further object of the present invention to provide a method for preparing attenuated IBDV mutants by introducing mutations in the IBDV genome in a controlled manner.
Moreover, it is an object of the present invention to provide a genetically engineered IBDV mutant comprising the appropriate amino acid residues which allow the mutant to grow